The invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination and a method for producing a site-specific DNA recombination. The invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medical research.
The use of site-specific DNA recombinases allows genetic manipulations in both prokaryotic and eukaryotic organisms. For this purpose, various site-specific DNA recombinases isolated from different organisms are used. The DNA recombination mediated by the site-specific recombinase occurs by cleavage and rejoining of DNA at specific DNA sequences, the so-called recognition sites (nucleic acid sequences of 10 to 150 base pairs). If two recognition sites are oriented in the same direction in a DNA strand, a nucleic acid segment flanked by the recognition sites is cut out (excision). If two recognition sequences flanking a nucleic acid segment in a DNA strand are oriented in the opposite direction, the site-specific DNA recombinase catalyzes the inversion of the nucleic acid segment. If two recombination sites are located on two different molecules, then the site-specific DNA recombinase catalyzes merge of two molecules (integration).
Among sites-specific recombinases a particular class called tyrosine recombinases (SSRs), such as Cre and Flp, has become an outstanding genetic tool. Unlike most SSRs, they do not require additional host factors for efficient catalysis and recognize relatively short sequences. Because of the simplicity and efficiency, these recombinases now serve as "molecular scissors" for robust, non-disruptive and reproducible genomic modifications.
The so-called Cre/loxP system (
An excerpt from the UniProt Database for CRE (Acc. No. FOV1A) discloses a putative Cre-like site-specific recombinase from Vibrio nigripulchritudo. However, only a small fraction of putative recombinase-like proteins is effective and belongs to the mentioned class of the recombinases with useful properties.
Another site-specific DNA recombinase system is the so-called Flp/FRT system isolated from Saccharomyces cerevisiae. The Flp/FRT system includes the recombinase Flp (flippase) (amino acid sequence according to SEQ ID No. 6) that catalyzes DNA-recombination on its recognition sites, the so-called FRT sites (SEQ ID No. 7).
In addition to the Cre/loxP and the Flp/FRT system, that are both the most widely used site-specific recombinase systems of tyrosine class, other recombinase systems are known in the art.
The known recombinase systems that are known in the art show different activities in cells of different origin. For many applications, such as the production of transgenic animals with conditional gene knockouts, two or more recombinase systems are used in combination with each other. However, emerging complex genetics studies and applications require simultaneous use of multiple recombinases. At the same time not all well-described site-specific recombinases are equally applicable in all model organisms due to, e.g. genome specificity (off-target activity on cryptic recognition target sites). For that reason it is important that an optimal recombinase can be chosen depending on the target organism or experimental setup. Therefore, there is a need for the provision of alternative recombinase systems that can be used to catalyze a site-specific DNA recombination on short targets in a variety of cell types and with high activity and with low toxicity.
It is the objective of the invention to provide novel recombinase systems for site-specific genetic recombination that can be used in a variety of cell types. Another object of the invention is to provide a novel, highly specific, recombinase system for site-specific genetic recombination with preferably low toxicity.
The invention is based on the finding that the protein YP_004250912.( http://www.ncbi.nlm.nih.gov/protein/YP_004250912.1, SEQ ID No. 37), which is referred to herein as Nigri (wild type), which was already predicted to be a putatice Cre-like recombinase, recognizes recognition sites according to SEQ ID No. 38. Nigri is derived from the gram-negative bacterium Vibrio nigripulchritudo. The recognition sequences for Cre and Nigri show a low identity of 34.7 % (34.7 % sequence similarity).
The inventors identified recognition sites for Nigri.
The objective is solved by the use of a protein with recombinase activity, wherein the protein comprises an amino acid sequence exhibiting at least 70 %, preferably at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % amino acid sequence identity to SEQ ID No. 37 to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other, wherein at least one recognition site comprises a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38; or a nucleic acid sequence that is a functional mutant thereof.
The protein with recombinase activity as defined above and used in the invention is referred to herein as "Nigri". The recognition site of the site-specific recombinase Nigri is referred to herein as "nox-site" or simply "nox". A nox-site is characterized by its nucleic acid sequence according to SEQ ID No. 38 or a nucleic acid sequence reverse complementary thereto. Recognition sites that exhibit a nucleic acid sequence identity to SEQ ID No. 38 of at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 %, or nucleic acid sequences reverse complementary thereto; and that are targets for the site specific recombination of Nigri (herein referred to as "functional mutants" of nox sites) are also "nox-sites" within the sense of the invention. However, particularly preferred nox-sites exhibit a nucleic acid sequence according to SEQ ID No. 38 or a nucleic acid sequence reverse complementary thereto (herein also referred to as "wild type nox-site").
By a functional mutant of the wild type nox-site is meant that one or more nucleic acids are added to, inserted, deleted or substituted from the nucleic acid sequence according to SEQ ID No. 38. At the same time, the functional mutant of the recognition site with a nucleic acid sequence according to SEQ ID No. 38 exhibits a nucleic acid sequence identity to SEQ ID No. 38 of at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % and is a functional recognition site of Nigri. Preferred mutations are point mutations or an exchange of the spacer region of the recognition site according to SEQ ID No. 38. It is known that the exchange of a spacer region of a recognition site does not influence its activity as a target site for the specific recombinase.
Within the sense of the invention the terms "site-specific DNA recombinase" and "recognition site" are used as defined above in the discussion of the prior art.
The invention further includes a method for producing a site-specific DNA recombination using Nigri. The method according to the invention comprises the steps of
- a) providing a cell comprising at least two recognition sites that are identical or reverse complementary to each other, wherein at least one recognition site comprises a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38; or a nucleic acid sequence that is a functional mutant thereof; and
- b) contacting a protein with recombinase activity, wherein the protein exhibits an amino acid sequence of at least 70 %, preferably at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % amino acid sequence identity to SEQ ID No. 37, with the recognition sites, thereby producing the site-specific DNA-recombination.
In this method according to the invention the Nigri protein is contacted with at least two nox sites inside a cell. Upon binding of the Nigri protein to the nox sites, site-specific DNA-recombination occurs.
The method according to the invention can be carried out in vitro or in vivo. In case the invention is carried out in an animal (including humans) it is preferably carried out for non-therapeutic use. The method is applicable in all areas where state of the art site specific recombinases are conventionally used (including inducible knock out or knock in mice and other non-human transgenic animal models). In preferred methods according to the invention, the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA. The method according to the invention is not used for the therapeutic treatment of a human being or an animal. Preferably the method according to the invention is used to create animal models, which are useful for biomedical research, e. g. as models for human diseases.
In a method according to the invention, the nucleic acid sequence encoding for Nigri is either already present in the cell or introduced into the cell, preferably by recombinant techniques. This preferred method according to the invention further includes the step of
- c) introducing into the cell a nucleic acid encoding for the Nigri protein with recombinase activity.
For activation of the expression of the nucleic acid encoding for Nigri, the nucleic acid encoding for Nigri further comprises a regulatory nucleic acid sequence, preferably a promoter region. Hence, expression of the nucleic acid encoding for the protein with recombinase activity is produced by activating the regulatory nucleic acid sequence. Accordingly, to induce a DNA recombination, the regulatory nucleic acid sequence (preferably the promoter region) is activated to express the gene encoding for the Nigri protein. Preferably, the regulatory nucleic acid sequence (preferably the promoter region) is either introduced into the cell in the method of the invention, preferably together with the sequence encoding for Nigri, or the regulatory nucleic acid sequence is already present in the cell in the beginning of the method according to the invention. In the second case, merely the nucleic acid encoding for the Nigri protein is introduced into the cell (and placed under the control of the regulatory nucleic acid sequence).
By the term "regulatory nucleic acid sequences" within the sense of the invention gene regulatory regions of DNA are meant. In addition to promoter regions the term encompasses operator regions more distant from the gene as well as nucleic acid sequences that influence the expression of a gene, such as cis-elements, enhancers or silencers. The term "promoter region" within the sense of the invention refers to a nucleotide sequence on the DNA allowing a regulated expression of a gene. In this case the promoter region allows regulated expression of the nucleic acid encoding for Nigri. The promoter region is located at the 5'-end of the gene and thus before the RNA coding region. Both, bacterial and eukaryotic promoters are applicable for the invention.
In a method according to the invention, the nox sites are either included in the cell or introduced into the cell, preferably by recombinant techniques. This preferred method according to the invention further includes the steps of introducing into a cell the following nucleic acids:
- a) a first nucleic acid (first recognition site, first nox site) comprising a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38; or a nucleic acid sequence that is a functional mutant thereof;
- b) a second nucleic acid (second recognition site, second nox site) comprising a nucleic acid sequence identical or reverse complementary to the nucleic acid sequence of the first nucleic acid (first recognition site).
In a preferred method according to the invention, a nucleic acid encoding for the Nigri protein and one or two, preferably two, nox sites, are introduced into the cell. This method includes the following steps:
- introducing into a cell the following nucleic acids:
- i) a nucleic acid encoding for Nigri, wherein the nucleic acid is introduced into the DNA such, that a regulatory nucleic acid sequence (preferably a promoter region) controls the expression of the nucleic acid encoding for Nigri,
- ii) a nucleic acid (first recognition site, first nox site) comprising a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38; or a nucleic acid sequence that is a functional mutant thereof;
- iii) a nucleic acid (second recognition site, second nox site) comprising a nucleic acid sequence identical or reverse complementary to the nucleic acid sequence defined in ii) (nucleic acid sequence of the first recognition site), and
- activating the regulatory nucleic acid sequence (preferably the promoter region) to induce expression of the first nucleic acid for the synthesis of the protein with recombinase activity.
By this preferred method according to the invention, the nucleic acid sequence encoding for Nigri , is introduced into a cell and at least two recognition sizes (nox sites) are introduced into the genomic or episomal DNA of the cell. The steps i) to iii) can be performed in arbitrary order.
The introduction of the nucleic acids into the cells is performed using techniques of genetic manipulation known by a person skilled in the art. Among suitable methods are cell transformation bacterial cells and transfection or viral infection for mammalian cells, whereby a nucleic acid sequence encoding the protein is introduced into the cell as a component of a vector or part of virus-encoding DNA or RNA. The cell culturing is carried out by methods known to a person skilled in the art for the culture of the respective cells. Therefore, cells are preferably transferred into a conventional culture medium, and cultured at temperatures (preferably 35-38 °C) and in a gas atmosphere that is conducive to the survival of the cells.
The method according to the invention can be performed using eukaryotic and prokaryotic cells; preferred prokaryotic cells are bacterial cells. Preferred prokaryotic cells are cells of Escherichia coli. Preferred eukaryotic cells are yeast cells (preferably Saccharomyces cerevisiae), insect cells, non-insect invertebrate cells, amphibian cells, or mammalian cells (preferably somatic or pluripotent stem cells, including embryonic stem cells and other pluripotent stem cells, like induced pluripotent stem cells, and other native cells or established cell lines, including NIH3T3, CHO, HeLa, HEK293, hiPS). In case of human embryonic stem cells, cells are obtained without destructing human embryos, e. g. by outgrowth of single blastomeres derived from blastocysts as described by (Chung 2008). Also preferred are cells of a non-human host organism, preferably non-human germ cells, somatic or pluripotent stem cells, including non-human embryonic stem cells, or blastocytes.
Further, the invention includes a nucleic acid comprising a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38, or a nucleic acid sequence that is a functional mutant thereof. A nucleic acid according to the invention comprises a maximum of 40, preferably 34, base pairs. This nucleic acid according to the invention includes the nox site of Nigri used according to the invention. Further the invention includes a vector (also referred to herein as "reporter vector") comprising at least one nucleic acid comprising a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38, or a nucleic acid sequence that is a functional mutant thereof. In a preferred embodiment of the invention the vector comprises at least two nox sites i.e. at least two nucleic acids that independently of each other exhibit a nucleic acid sequence according to or reverse complementary to SEQ ID No. 38, or a nucleic acid sequence that is a functional mutant thereof. The at least two nox sites are positioned such that they are flanking a DNA segment, that upon recognition of the nox sites by Nigri, the DNA segment is either excised or inverted. Thereby the DNA segment can preferably contain a gene or a promoter region. As described above, the DNA segment is excised when it is flanked by two sites of the same orientation (same nucleic acid sequence). An inversion of the DNA segment is catalyzed by Nigri, when the DNA segment is flanked by two nox sites, arranged in opposite orientations (i.e. comprise a nucleic acid sequence reverse complementary to one another).
The term "nucleic acids" as used herein includes not only deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), but also all other linear polymers in which the bases adenine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U) are arranged in a corresponding sequence (nucleic acid sequence). The invention also comprises the corresponding RNA sequences (in which thymine is replaced by uracil), complementary sequences and sequences with modified nucleic acid backbone or 3 'or 5'-terminus. Nucleic acids in the form of DNA are however preferred.
The term reporter vector as used herein includes a plasmid, virus or other nucleic acid carriers, that comprise a nucleic acid sequence according to the invention by genetic recombination (recombinantly), e.g. by insertion or incorporation of said nucleic acid sequence. Prokaryotic vectors as well as eukaryotic vectors, for example artificial chromosomes, such as YAC (yeast artificial chromosomes), are applicable for the invention. Typically, the expression vector comprises an origin of replication, a promoter, as well as specific gene sequences that allow phenotypic selection of host cells comprising the reporter vector.
The invention also includes a nucleic acid that encodes for a protein with recombinase activity, , wherein the protein preferably comprises an amino acid sequence exhibiting at least 70%, preferably at least 80%, preferably at least 90%, particularly preferred at least 95%, even more preferred at least 99% amino acid sequence identity to SEQ ID No. 37. Particularly preferred is a nucleic acid encoding for Nigri, used according to the invention with a nucleic acid sequence according to SEQ ID 37. Furthermore, the invention includes a vector comprising said nucleic acid according to the invention (encoding for the protein with recombinase activity).
The invention also includes the use of the nucleic acids or vectors according to the invention in a method according to the invention for producing a site-specific DNA recombination.
When a nucleic acid encoding for a Nigri protein, in particular in the form of a vector according to the invention, and at least two nox-sites as recognition sites are introduced into a host cell, a site-specific recombination is catalyzed upon expression of the protein Nigri by its recognition of the nox sites.
Accordingly, the invention includes an isolated host cell comprising the following recombinant DNA fragments:
- at least one, preferably at least two, nucleic acids according to the invention comprising a nox-site (preferably two nucleic acids according to the invention that include a nox-site, respectively, flank a further DNA segment) and/or a nucleic acid according to the invention encoding for a Nigri protein or
- a vector according to the invention comprising at least two nucleic acids comprising a nox-site (preferably two nucleic acids according to the invention that include a nox-site, respectively, flank a further DNA segment) and/or a vector according to the invention comprising a nucleic acid encoding for a Nigri-protein.
The invention concerns only those isolated host cells that comprise the above mentioned nucleic acids or vectors recombinantly and not naturally, i.e. by genetic modification of the host cell. In particular the invention does not include cells of the organism Pantoea sp. aB or Vibrio nigripulchritudo that contain a nucleic acid sequence encoding for Nigri and comprising the recognition site nox.
Particularly preferred are isolated host cells that contain both, a nucleic acid encoding for Nigri and at least two nox-sites (which are either oriented in the same or in opposite direction).
A host cell within the sense of the invention is a naturally occurring cell or a (optionally transformed or genetically modified) cell line that comprises at least one vector according to the invention or a nucleic acid according to the invention recombinantly, as described above. Thereby, the invention includes transient transfectants (e.g. by mRNA injection) or host cells that include at least one expression vector according to the invention as a plasmid or artificial chromosome, as well as host cells in which an expression vector according to the invention is stably integrated into the genome of said host cell. The host cell is preferably selected from cells of prokaryotes and eukaryotes. Preferred prokaryotic cells are cells of Escherichia coli. Preferred eukaryotic cells are selected from yeast cells (preferably Saccharomyces cerevisiae), insect cells, non-insect invertebrate cells, amphibian cells, and mammalian cells (preferably embryonal stem cells, NIH3T3, CHO, HeLa, HEK293, hiPS). Embryonal stem cells derived by killing of human embryos are specifically excluded from the term "host cells" within the sense of the invention.
With the invention it is also possible to induce tissue-specific site-specific recombination in non-human host organisms, such as mammals. Therefore the invention also includes a non-human host organism comprising the following recombinant DNA fragments:
- at least one, preferably at least two, nucleic acids according to the invention comprising a nox-site (preferably two nucleic acids according to the invention that include a nox-site , , flank a further DNA segment) and/or a nucleic acid according to the invention encoding for a Nigri-protein or
- a vector according to the invention comprising at least two nucleic acids comprising a nox-site (preferably two nucleic acids according to the invention that include a nox-site, , flank a further DNA segment) and/or a vector according to the invention comprising a nucleic acid encoding for a Nigri-protein.
Explicitly included are non-human host organisms that only comprise a recombinant nucleic acid encoding for a Nigri protein (and that do not comprise a nucleic acid including a nox site). Furthermore, the invention includes non-human host organisms that only comprise at least one, preferably at least two, nox-sites (and that do not comprise a nucleic acid encoding for a Nigri protein). In that case, at least two nox sites preferably flank another DNA segment. Upon cross-breeding of two non-human host organisms, wherein a first host organism comprises a recombinant nucleic acid encoding for a Nigri protein and a second host organism comprises at least two recombinant nox sites and preferably flanking a further DNA segment, the offspring includes host organisms expressing Nigri and further including the recognition sites nox, so that a site-specific DNA-recombination, like a tissue-specific conditional knock-out, is possible.
Non-human host organisms comprise a vector according to the invention or a nucleic acid according to the invention as described above that is, respectively, stably integrated into the genome of the host organism or individual cells of the host organism. Preferred host organisms are plants, invertebrates and vertebrates, particularly Bovidae, Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, medaka, zebrafish, or Mus musculus, or embryos of these organisms.
The invention also includes a method for providing a non-human host organism, comprising the following steps:
- providing a first non-human host organism comprising a nucleic acid encoding for a Nigri protein,
- providing a second non-human host organism comprising at least two nucleic acids, the nucleic acid sequence of which is independently from each other comprising a nox site (a nucleic acid sequence according to SEQ ID No. 38) or a nucleic acid sequence reverse complementary thereto or a nucleic acid sequence that is a functional mutant of the aforementioned nucleic acid sequences, wherein the at least two nucleic acids preferably flank another DNA segment (in particular a gene or a promoter region),
- cross-breeding of the first and the second non-human host organism and from the offspring obtained thereby selecting the non-human host organisms that comprise a nucleic acid encoding for a Nigri protein and at least two nox sites (in case of Nigri, the nucleic acid sequence of which is independently from each other comprising a nucleic acid sequence according to SEQ ID No. 38 or a nucleic acid sequence reverse complementary thereto or a nucleic acid sequence that is a functional mutant of the aforementioned nucleic acid sequences).
The invention provides a novel recombinase system suitable for producing a site-specific recombination in cells of various cell types. Thereby, a diverse range of genetic manipulations can be realized, particularly rearrangements of the DNA fragments flanked by nox sites in same orientation (excision), opposite orientation (inversion) or when one nox site is present on each of two DNA molecules with one if it being in circular form in any orientation (integration). Exemplary manipulations are the excision of a DNA segment that is flanked by two nox sites oriented in the same direction mediated by the Nigri recombinase. Amongst others, the recombinase systems according to the invention provides the possibility to excise a nox-flanked stopper DNA fragment which is located 5' of the gene and 3' of the corresponding to the gene promoter. Without recombination the stopper sequence prevents gene expression, whereas upon recombinase-mediated excision of the stopper via two flanking recognition sites the gene is located to the proximity of the promoter and therefore is expressed. Different types of promoter regions that regulate the expression of the recombinase allow, inter alia, conditional DNA recombination, when for example a tissue or organism-specific or inducible promoter region is used to express the recombinase.
For the recombinase systems according to the invention no cross-reactivity with other recombinase systems was observed. Therefore, the recombinase systems according to the invention are applicable for use in combination with other recombinase systems and become a particular valuable tool for genetic experiments where multiple recombinases are required simultaneously or sequentially. In bacterial cells, in particular in E. coli, a similar level of activity compared to the Cre/loxP system could be demonstrated. In E. coli, the recombinase systems according to the invention showed a significantly higher activity than the VCre/VloxP system. In addition, the recombinase system according to the invention is highly effective and specific in mammalian cells as well.
Due to its efficiency in a variety of cell types the recombinase systems according to the invention can be used in particular for producing site-specific DNA recombination in cells in which other recombinase systems were shown to only achieve poor results. For example, one of the most widely used recombinase system Cre/loxP has limited application in some model organisms such as Caenorhabditis elegans, supposedly due to the presence of loxP-like nucleic acid sequences being naturally present in the genome of C. elegans.
The object of the invention is also solved by the use of a protein with recombinase activity, wherein the protein comprises an amino acid sequence exhibiting at least 70 %, preferably at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % amino acid sequence identity to one of the amino acid sequences according to SEQ ID No. 37 to catalyze a site-specific DNA recombination. The aforementioned site-specific recombinase is used for site-specific DNA recombination at, preferably two, recognition sites that are identical or reverse complementary to each other, wherein at least one recognition site comprises a nucleic acid sequence according to or reverse complementary to the nucleic acid sequence according to SEQ ID No.38, respectively; or a nucleic acid sequence that is a functional mutant thereof.
The proteins with recombinase activity and recognition sites are used in the following combination (table 1). Derivatives of the respective protein with amino acid sequence identities of at least 70 %, preferably at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % and recognition sites with a nucleic acid sequence that is reverse complementary to the indicates nucleic acid sequence as well as their functional mutants are also included. It is preferred to use the indicated proteins with recombinase activity on wild type recognition sites or recognition sites with a nucleic acid sequence reverse complementary thereto.
The invention is further based on the finding of the inventors that each of the proteins listed in table 1 in column 2, shows a Cre recombinase-like activity and recognizes recognition sites as indicated in table 1 in column 3. The organisms from which each of the site-specific recombinases is derived from are also indicated in table 1 (column 1). All of the proteins with recombinase activity listed in table 1 show a low amino acid sequence identity to Cre recombinase.
The invention further relates to a method for producing a site-specific DNA-recombination by contacting a protein with recombinase activity with the indicated recognition sites.
The invention also includes nucleic acids exhibiting a length not more than 40 base pairs, each nucleic acid comprising a nucleic acid sequence according to SEQ ID No. 38, a nucleic acid sequence that is a functional mutant thereof or a nucleic acid sequence reverse complementary thereto.
Additionally, the invention relates to vectors comprising said nucleic acid sequences, preferably at least one, even more preferred at least two identical or reverse complementary nucleic acid sequences.
The use of any of the vectors according to the invention in a method for producing a site-specific DNA-recombination is also included in the invention.
Further, the invention includes an isolated host cell or an non-human isolated host organism comprising
- at least one, preferably at least two, nucleic acids according to the invention comprising a recognition site as defined above (preferably two nucleic acids according to the invention that include a recognition site, respectively, flank a further DNA segment) and/or a nucleic acid according to the invention encoding for a protein with recombinase activity, as defined above, or
- a vector according to the invention comprising at least two nucleic acids comprising a recognition site as defined above (preferably two nucleic acids according to the invention that include a recognition site, respectively, flank a further DNA segment) and/or a vector according to the invention comprising a nucleic acid encoding for a protein with recombinase activity as defined above.
- whereas isolated host cells that comprise the nucleic acids or the vectors naturally and cells that are obtained by the destructing of human embryos are excluded.
The embodiments of the invention that are described above in detail for site-specific recombination with Nigri on nox sites are also included within the invention for site-specific recombination with one of the above mentioned site-specific recombinases on their recognition sites.
For each of the proteins with recombinase activity, the inventors identified numerous putative recognition sites with a lox-like structure in the organism wherefrom the recombinase was derived. Upon extensive studies only one thereof, the indicated respective wild type recognition site, turned out to be the actual recognition site of the indicated protein with recombinase activity. By expression of the nucleic acid sequence encoding for the site-specific recombinase in E. coli, the recombinase protein could be successfully obtained and its recombinase activity and specificity for the respective wild type recognition site could be demonstrated. It was shown that the indicated proteins with recombinase activity do not cross react with other known recombinase systems and are superior in the activity compared to some of the known recombinase systems, at least in certain cell types.
The invention is further illustrated by the following figures and examples without being limited to these.
To verify whether the protein Panto and Nigri exhibit recombinase activity and to verify that pox and nox are corresponding recognition sites, a nucleic acid encoding for Nigiri was cloned into an E. coli recombination reporter plasmid that comprised two nox-sites of the same orientation. A plasmid containing gene coding for Panto protein and two identical sequences of pox site was created in a similar way. The recombination reporter plasmid was based on the plasmid pEVO (Buchholz and Stewart, 2001), in which the recombinase was inserted via a BsrGI and XbaI cleavage site. In the plasmid, the two vox-sites flanked an approximately 1kb DNA segment that was excised by DNA-recombination (Fig. 12). DNA recombination was induced by the addition of L (+)-arabinose. It was shown that Vika mediated a DNA recombination at pox-sites Panto and Nigri on nox sites. Therefore, it could be shown that the Panto/pox and Nigri/nox system are recombinase systems applicable in E. coli cells.
Buchholz, F., and Bishop, J. (2001). loxP-directed cloning: Use Cre recombinase as a universal restriction enzyme. BioTechniques 31, 906 Buchholz, F., and Stewart, A.F. (2001). Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol 19, 1047-1052 Chung Y et al. Cell Stem Cell 2008 (2): 113 - 117 Lin et al. Cell Res 2007; 17:999-1007 Mai et al. Cell Res 2007 17:1008-1019 Suzuki, E., and Nakayama, M. (2011). VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering. Nucleic Acids Research 39, e49-e49
- <110> Technische Universität Dresden
- <120> Protein with recombinase activity for site-specific
DNA-recombination - <130> 00017P0181EPWO
- <150>
EP 12 177 608.2
<151> 2012-07-24 - <160> 38
- <170> PatentIn version 3.5
- <210> 1
<211> 361
<212> PRT
<213> Vibrio coralliilyticus - <400> 1
- <210> 2
<211> 34
<212> DNA
<213> Vibrio coralliilyticus - <400> 2
aataggtctg agaacgccca ttctcagacg tatt 34 - <210> 3
<211> 1083
<212> DNA
<213> Vibrio coralliilyticus - <400> 3
- <210> 4
<211> 343
<212> PRT
<213> Bacteriophage P1 - <400> 4
- <210> 5
<211> 34
<212> DNA
<213> Bacteriophage P1 - <400> 5
ataacttcgt atagcataca ttatacgaag ttat 34 - <210> 6
<211> 423
<212> PRT
<213> Saccharomyces cerevisiae - <400> 6
- <210> 7
<211> 34
<212> DNA
<213> Saccharomyces cerevisiae - <400> 7
gaagttccta tactttctag agaataggaa cttc 34 - <210> 8
<211> 342
<212> PRT
<213> Enterobacteria phage D6 - <400> 8
- <210> 9
<211> 32
<212> DNA
<213> Eubacterium xylanophilum - <400> 9
taactttaaa taattggcat tatttaaagt ta 32 - <210> 10
<211> 380
<212> PRT
<213> Vibrio plasmid p0908 - <400> 10
- <210> 11
<211> 34
<212> DNA
<213> Vibrio plasmid p0908 - <400> 11
tcaatttccg agaatgacag ttctcagaaa ttga 34 - <210> 12
<211> 34
<212> DNA
<213> Homo sapiens - <400> 12
cagagctctg agactttgtg ttctcaaaga tatc 34 - <210> 13
<211> 34
<212> DNA
<213> Homo sapiens - <400> 13
aagaggtgtg agactgaatt ttctcagtca ggtt 34 - <210> 14
<211> 34
<212> DNA
<213> Homo sapiens - <400> 14
aagagaactg agaaaatatt ctctcagagg gaat 34 - <210> 15
<211> 34
<212> DNA
<213> Mus musculus - <400> 15
aacagctttg agagctgttg ctctcagctg aatt 34 - <210> 16
<211> 34
<212> DNA
<213> Homo sapiens - <400> 16
aacaggactg agataaaaca gtctcagaca gcat 34 - <210> 17
<211> 34
<212> DNA
<213> Homo sapiens - <400> 17
acaaccattt ataatatata atatatgatg ttat 34 - <210> 18
<211> 34
<212> DNA
<213> Mus musculus - <400> 18
gtaactgagt atatgcatat atatacgtat atat 34 - <210> 19
<211> 366
<212> PRT
<213> Vibrio tubiashii ATCC 19109 - <400> 19
- <210> 20
<211> 34
<212> DNA
<213> Vibrio tubiashii ATCC 19109 - <400> 20
catacgtcct agaatggcag ttctaggacg tatt 34 - <210> 21
<211> 353
<212> PRT
<213> Methylobacterium extorquens DM4 - <400> 21
- <210> 22
<211> 34
<212> DNA
<213> Methylobacterium extorquens DM4 - <400> 22
atttcccgcg atagatggtg ttatcgcagg caat 34 - <210> 23
<211> 386
<212> PRT
<213> Streptomyces sp. W9 - <400> 23
- <210> 24
<211> 34
<212> DNA
<213> Streptomyces sp. W9 - <400> 24
gttgcccccg tcgcgcggtc gcgttggggg caac 34 - <210> 25
<211> 360
<212> PRT
<213> Streptomyces sp. SPB74 - <400> 25
- <210> 26
<211> 34
<212> DNA
<213> Streptomyces sp. SPB74 - <400> 26
ctggctcttg gtaaggcacg ttatcaagag ccaa 34 - <210> 27
<211> 346
<212> PRT
<213> Agrobacterium tumefaciens str. C58 plasmid At - <400> 27
- <210> 28
<211> 34
<212> DNA
<213> Agrobacterium tumefaciens str. C58 plasmid At - <400> 28
agccatcaag atggcagacg ccatcttgat ggct 34 - <210> 29
<211> 405
<212> PRT
<213> Chelativorans sp_ BNC1 plasmid 3 - <400> 29
- <210> 30
<211> 34
<212> DNA
<213> Chelativorans sp_ BNC1 plasmid 3 - <400> 30
acatcgagcg gctccgcgac gaaccgcgcg atgt 34 - <210> 31
<211> 347
<212> PRT
<213> Marinobacter aquaeolei VT8 plasmid pMAQU02 - <400> 31
- <210> 32
<211> 34
<212> DNA
<213> Marinobacter aquaeolei VT8 plasmid pMAQU02 - <400> 32
ctaacccacg ataatcaatc ttatcgcggg ttaa 34 - <210> 33
<211> 351
<212> PRT
<213> Pseudomonas sp. ND6 plasmid pND6-1 - <400> 33
- <210> 34
<211> 34
<212> DNA
<213> Pseudomonas sp. ND6 plasmid pND6-1 - <400> 34
ttgacccacg ataagcgcgg ttatcgtgag ttaa 34 - <210> 35
<211> 343
<212> PRT
<213> Pantoea sp. aB - <400> 35
- <210> 36
<211> 34
<212> DNA
<213> Pantoea sp. aB - <400> 36
gaaactttaa ataataagtc ttatataaag tttc 34 - <210> 37
<211> 460
<212> PRT
<213> Vibrio nigripulchritudo - <400> 37
- <210> 38
<211> 34
<212> DNA
<213> Vibrio nigripulchritudo - <400> 38
tgaatgtcct ataattacac ttataggaca ttca 34